G-protein coupled receptors (GPCRs) are proteins responsible for transducing a signal within a cell. GPCRs have usually seven transmembrane domains. Upon binding of a ligand to an extra-cellular portion or fragment of a GPCR, a signal is transduced within the cell that results in a change in a biological or physiological property or behaviour of the cell. GPCRs, along with G-proteins and effectors (intracellular enzymes and channels modulated by G-proteins), are the components of a modular signaling system that connects the state of intra-cellular second messengers to extra-cellular inputs.
GPCR genes and gene products can modulate various physiological processes and are potential causative agents of disease. The GPCRs seem to be of critical importance to both the central nervous system and peripheral physiological processes.
The GPCR protein superfamily is represented in five families: Family I, receptors typified by rhodopsin and the beta2-adrenergic receptor and currently represented by over 200 unique members; Family II, the parathyroid hormone/calcitonin/secretin receptor family; Family III, the metabotropic glutamate receptor family, Family IV, the CAMP receptor family, important in the chemotaxis and development of D. discoideum; and Family V, the fungal mating pheromone receptor such as STE2.
G proteins represent a family of heterotrimeric proteins composed of α, β and γ subunits, that bind guanine nucleotides. These proteins are usually linked to cell surface receptors (receptors containing seven transmembrane domains) for signal transduction. Indeed, following ligand binding to the GPCR, a conformational change is transmitted to the G protein, which causes the α-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the βγ-subunits.
The GTP-bound form of the α, β and γ-subunits typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cAMP (e.g. by activation of adenyl cyclase), diacylglycerol or inositol phosphates.
Greater than 20 different types of α-subunits are known in humans. These subunits associate with a small pool of β and γ subunits. Examples of mammalian G proteins include Gi, Go, Gq, Gs, G-olf and Gt. G proteins are described extensively in Lodish et al., Molecular Cell Biology (Scientific American Books Inc., New York, N.Y., 1995; and also by Downes and Gautam, 1999, The G-Protein Subunit Gene Families. Genomics 62:544-552), the contents of both of which are incorporated herein by reference.
Known and uncharacterized GPCRs currently constitute major targets for drug action and development. For some GPCRs a possible physiological role has been assigned, however no ligands capable of modulating said receptor have been identified yet. There are ongoing efforts to identify new G protein coupled receptors which can be used to screen for new agonists and antagonists having potential prophylactic and therapeutical properties.
More than 300 GPCRs have been cloned to date. Mechanistically, approximately 50-60% of all clinically relevant drugs act by modulating the functions of various GPCRs (Cudermann et al., J. Mol. Med., 73:51-63, 1995).
The sense of smell allows chemical communications between living organisms from invertebrates to mammals and environment. Perception and discrimination of thousands of odorants is made through olfaction. Such chemical signalling may modulate social behaviour of most animal species which rely on odorant compounds to identify kin or mate, to locate food or to recognize territory for instance. Smelling abilities are initially determined by neurons in the olfactory epithelium, the olfactory sensory neurons (OSN). Therein, odorant molecules bind to olfactory receptor proteins (OR), also known as odorant receptors. These OR are members of the G-protein coupled receptors (GPCR) superfamily. They are encoded by the largest gene family. While in rodents as many as 1,300 different OR genes have been identified, around 800 OR genes have been identified in the human genome. Each olfactory neuron is thought to express only one type of OR, forming therefore cellular basis of odorant discrimination by olfactory neurons. They are synthesized in the endoplasmatic reticulum, transported and eventually concentrated at the cell surface membrane of the cilia at the tip of the dendrite. Similarly, ORs are found at the axon terminal of OSN. They are assumed to play a role in targeting axons to OR-specific olfactory bulb areas.
Most mammals have a secondary olfactory system, the vomeronasal system. The vomeronasal organ is localized in nasal cavity and is partly made of vomeronasal sensory neurons. This system would be responsible for detecting pheromones through activation of pheromone receptors. However, there is no evidence to affirm that detection of pheromone is solely done through vomeronasal sensory neurons and that vomeronasal sensory neurons detect pheromone only. Pheromone receptors are also 7TM proteins, but they are completely distinct from the OR superfamily. Even though pheromone receptors are part of the GPCR superfamily, no G-protein coupled to those receptors has been identified yet. Two families of pheromone receptors have been listed to date: the V1R and the V2R families. Receptors of both of them have been identified in mouse (more than 300) while only 5 receptors of the V1R family in human.
Taste is also part of chemosensation. It relies on the activation of taste receptors localized on the tongue and palate in human. They are expressed in taste receptor cells (TCRs) part of taste buds. These cells are specialized epithelium cells that contact neurons, which in turn relay the information to the brain. Thereby, unlike OSN, TCRs are not neuron cells. As olfactory receptors, taste receptors are part of the GPCR superfamily. Today, 2 families have been identified: T1R and T2R families. While human T1R family is made of three receptors namely T1R1, T1R2 and T1R3, T2R family is made of 25 putative receptors in human. T2R receptors are responsible for bitter taste detection and would be functional as monomers. However, T1R receptors are thought to work as dimmers. Dimerization would confer specificity to receptors. Heterodimers of T1R1/T1R3 detect umami taste, while T1R2/T1R3 heterodimers are activated by sweet compounds. Besides those two families, other proteins are thought to be taste receptors such as TRMP5, a potential channel, mGluR4 that might function as an umami receptor, ASIC2, a sour taste receptor, ENaC, a salt taste receptor, VN1, a burning taste receptor or TMP8, a cold taste receptor.
Smell, taste and pheromones constantly influence personal behaviour of animals and humans. It is thus of great importance to understand mechanisms of said perceptions. Most particularly to determine means to influence it. Already known is that olfactory, taste and pheromone systems do not follow the one ligand/one receptor rules. Several ligands have been described in the literature to activate same receptors. Therefore said sensory systems are probably part of a system wherein different receptors may be activated by same ligands, and wherein one receptor may be modulated by different ligands.
RCC356, also termed PHOR-1, has previously been characterized and described as a novel prostate-specific GPCR upregulated in prostate cancer (U.S. Pat. No. 6,790,631 and US2004/0248088). The human polynucleotide sequence of RCC356 is shown in FIG. 1a; the human amino acid sequence of RCC356 in FIG. 1b. Said amino acid sequence shows one or more GPCR signature sequences and olfactory receptor signatures. RCC356 can thereby be considered as an olfactory receptor (OR). That this OR may have a prostate specific function is not exceptional. Indeed, OR genes were found to be expressed in tissues other than the olfactory epithelium, indicating potential alternative biological roles of this class of chemosensory receptors. In particular, it has been previously shown that ORs, other than RCC356, are also expressed in germ cells, testis, insuline-secreting β-cells, spleen, specific brain areas and heart (Parmentier et al. 1992, Nature, 355: 453-455; Thomas et al. 1996, Gene, 178:1-5; Nef and Nef 1997, Proc., Natl. Acad. Sci. USA, 94: 4766-4771; Blache et al. 1998, Biochem. Biophys. Res. Commun., 142 :669-672; Drutel et al, 1995, Receptors Channels 3:33-40; Ferrand et al. 1999, J. Mol. Cell. Cardiol. 31:1137-1142; Raming et al., 1998, Receptors Channels 6 :141-151). Furthermore, a rat olfactory receptor expressed in brain, known as RA1c (Raming et al., 1998, Receptor Channels 6:141), has a sequence with the highest degree of homology to PHOR-01. PHOR-1 is 59.9% identical to RA1c in 299 residue overlap. The likely human homologue of RA1c, HPRAAJ70, also shows a similar degree of homology to PHOR-01. The HPRAAJ70 protein is reported to be a prostate-specific GPCR (U.S. Pat. No. 5,756,309, WO 96/39435) confirming the finding mentioned in U.S. Pat. No. 6,790,631. U.S. Pat. No. 6,790,631 also mentiones that PHOR-01 is restricted to normal prostate, as well as to cancers of the prostate, kidney, uterus, cervix, stomac, and rectum. PHOR-01 may also be expressed in other cancers. Their role in the regulation of cell proliferation and transformation has also been suggested. For instance and as shown in U.S. Pat. No. 6,790,631, PHOR1 may play a critical role in cell proliferation. In this context, U.S. Pat. No. 6,790,631 proposes to use PHOR-01 in diagnostic and therapeutic methods and compositions useful in the management of various cancers that express PHOR-01, in particular of prostate cancers. Although a potential physiological role has been assigned to RCC356, reducing the doubling time of cells overexpressing RCC356 (U.S. Pat. No. 6,790,631), there is until now no indication by which ligand said receptor may be modulated.
Isovalaric acid is an unpleasant smelling organic acid forming part of the malodour formation of human and animal secretions, particular of sweat. Another constituent of human sweat is 3-methyl-2-hexenoic acid, of foul malodour is propionic acid, and of pet malodor is hexenoic acid. Said compounds were previously described as being recognized by a subgenus of olfactory receptors (US2003/0207337). Olfactory receptors belong to the 7-transmembrane receptor superfamily (Buck et al., Cell 65:175-87, 1991) which is known as G-protein coupled receptors (GPCRs). GPCRs mediate transmembrane signalling which controls many physiological functions, such as endocrine function, exocrine function, heart rate, lipolysis, carbohydrate metabolism, neurotransmission, vision, and taste reception. The olfactory receptors specifically recognize molecules that elicit specific olfactory sensation. These molecules are also referred to as ‘odorants’. Genes coding for the olfactory receptors are active primarily in olfactory neurons (Axel, Sci. Amer. 273:154-59, 1995). Individual olfactory receptor subtypes are expressed in subsets of cells distributed in distinct zones of the olfactory epithelium (Breer, Semin. Cell Biol., 5:25-32, 1994). But, as mentioned above, expression of OR is not limited to olfactory epithelium. Many laboratories have evidenced expression of OR in other tissues including prostate, brain, lung, liver, kidney, cervix, and breast.